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Article Cite This: J. Org. Chem. 2018, 83, 194−202

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Determination of the Absolute Configuration of Polyhydroxy Compound Ostreol B Isolated from the Dinoflagellate Ostreopsis cf. ovata Buyng Su Hwang,† Eun Young Yoon,‡ Eun Ju Jeong,§ Jaeyeon Park,‡ Eun-Hee Kim,∥ and Jung-Rae Rho*,† †

Department of Marine Biotechnology, Kunsan National University, 558 Daehak-ro, Gunsan 54150, South Korea Environment and Resource Convergence Center, Advanced Institutes of Convergence Technology, Suwon 16229, Republic of Korea § Department of Agronomy & Medicinal Plant Resources, Gyeongnam National University of Science and Technology, JinJu 52725, South Korea ∥ Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chungbuk 28119, Korea ‡

S Supporting Information *

ABSTRACT: Following isolation of the polyhydroxy compound, ostreol B, from cultivated cells of the toxic dinoflagellate Ostreopsis cf. ovata collected in South Korea, 1D and 2D NMR spectroscopy were employed to determine the planar chemical structure of this compound, which contained a tetrahydropyran ring, two terminal double bonds, and 21 hydroxyl groups. The absolute configurations of all stereogenic carbon centers in ostreol B were then determined through a combination of the J-based configuration analysis, rotating frame Overhauser effect correlations, and the modified Mosher method following cleavage of the 1,2-diol bonds. Ostreol B was also found to exhibit moderate cytotoxicity in HepG2, Neuro-2a and HCT-116 cells.

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remain undetermined due to the flexibility of such chains. However, despite the absence of rigid skeletons, the absolute configuration of a number of linear chain compounds, such as amphidinol-3 and kalotoxin-2, has been successfully determined by a combination of diverse NMR experiments and chemical reactions.13,14 Thus, following its isolation from Ostreopsis cf. ovata, we herein attempt to establish the absolute configuration of ostreol B (1, Scheme 1) through extensive J-based configuration analysis (JCBA) and the modified Mosher method using high field NMR systems.

INTRODUCTION The benthic dinoflagellates have received growing attention in recent years due to their toxic nature, which has been responsible for damage to human health, fisheries, and aquaculture. Among the known benthic dinoflagellates, the Ostreopsis species isolated from the Mediterranean Sea has invoked increasing interests worldwide following its recognition as a producer of ovatoxins and other PLTX analogues.1−4 The bloom of this species is also known to cause intoxication in human through aerosols.5,6 Thus, due to the appearance of O. cf. ovata in the waters of Korea, the exploration of toxic materials isolated from this species was achieved through the cultivation of O. cf. ovata from single cells collected from Jeju Island, South Korea. Indeed, we recently isolated the cytotoxic polyhydroxy compound, ostreol A, from the cultured extract, the structure of which is characterized by the presence of polyol functionalities attached to a long carbon backbone.7 The search for additional cytotoxic compounds from this extract provided a further compound bearing a similar polyhydroxy functionality on a long carbon backbone, namely ostreol B. Unlike ostreol A, this compound is composed of a tetrahydropyran ring and two long chains terminated by the exo-methylene group. In addition, this chain contains several 1,2-diol, 1,3-diol, and 1,3,5-triol units, which are commonly found in toxic compounds isolated from dinoflagellates.8−12 In many cases, the configurations of the hydroxyl groups within this long chain © 2017 American Chemical Society

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RESULTS AND DISCUSSION Ostreol B (1) was isolated as a white solid from a fraction containing both ostreol A and ostreol B and was assigned the molecular formula C57H108O22 based on high-resolution Scheme 1. Structure of Ostreol B (1)

Received: October 10, 2017 Published: November 29, 2017 194

DOI: 10.1021/acs.joc.7b02569 J. Org. Chem. 2018, 83, 194−202

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The Journal of Organic Chemistry Table 1. 1H and 13C NMR Data for Ostreol B (1)a

a

Data obtained on a Bruker 900 MHz instrument in CD3OD. Where, s = C; d = CH; t = CH2; and q = CH3.

(including 23 oxymethine groups), 28 methylene moieties, and one methyl group. Further elucidation of the chemical structure of ostreol B (1) was carried out by the examination of 1H−1H double-quantum filtered homonuclear correlation spectroscopy (DQF-COSY) correlations using the well-resolved oxymethine protons or upfield protons [δH 3.78 (H-10), 1.14 (H-12), 3.63(H-19), 1.38(H-22), 3.75(H-26), 3.27(H-28), 4.15(H-29), 4.01(H-31), 3.23(H-34), 3.28(H-38), 3.68(H-41), 3.52(H-46), and 0.91(H57)]. Interpretation of the DQF-COSY and total correlation spectroscopy (TOCSY) spectra gave the following connectivity, which is indicated in bold in Figure 1: C-1 to C-2, C-10 to C14, C-18 to C-23, C-25 to C-35, C-37 to C- 43, C-45 to C-47, and C-52 to C-56. These correlations were also verified by 1 H−13C HSQC-TOCSY correlations. In addition, the assignment of C-24 was achieved through the observation of mutual HSQC-TOCSY correlations with H-22 and H-26. Similarly, C44 was also assigned through common HSQC-TOCSY

electrospray ionization time-of-flight mass spectrometry (HRESI-TOF MS) ([M + Na]+ = 1167.7227, Δ = 0.3). This formula was consistent with four degrees of unsaturation. In addition, the infrared (IR) and ultraviolet (UV) spectra indicated the presence of hydroxyl groups (3343 cm−1) and conjugated dienes (235 nm ε 19400), respectively. Furthermore, the 1H NMR spectrum exhibited signals corresponding to numerous oxymethine moieties, four olefinic signals, two exo-methylene groups, one methyl group, and severely overlapped resonances between 1.2 and 2.3 ppm that correspond to saturated hydrocarbon chains. However, although the 13C NMR spectrum suffered from extensive signal degeneracy, four signals corresponding to sp2 carbon atoms and two signals corresponding to exo-methylene moieties were clearly identified based on their typical chemical shifts (Table 1). A 1H−13C multiplicity-edited heteronuclear single quantum correlation (HSQC) experiment identified 28 methine groups 195


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The Journal of Organic Chemistry

carbon atoms, and C-6. Similarly, methylene proton H-52, which was coupled with a further olefinic proton, gave HSQCTOCSY correlations with two olefinic carbon atoms and a number of overlapped carbon signals in the aliphatic region. Moreover, an HMBC correlation between H-24 and C-28 confirmed that the remaining cyclic moiety was a tetrahydropyran ring with an ether linkage between C-24 and C-28, thereby completing the planar structure of 1. Indeed, this structure was confirmed by MS/MS fragmentation of 1, as outlined in Figure S2. Additionally, following cleavage of the 1,2-diol bonds of 1 by treatment with NaIO4 and NaBH4, signals at m/z 385 [M + Na]+ corresponding to fragment I (2) and at 263 [M + Na]+ corresponding to fragment II (3) confirmed the carbon chain lengths at the terminal parts (Figure 2). Following confirmation that ostreol B (1) contains 24 stereogenic centers on a long carbon chain, which consists of a tetrahydropyran ring in the middle and each exo-methylene group on both termini, we proposed determination of the configuration of all stereogenic centers through a combination of the modified Mosher method, extensive JBCA,15 and ROE correlations, comparison of the corresponding carbon chemical shifts with Kishi’s Universal NMR database, and using the empirical rule for 1,5-diol systems reported by Matsunaga et al.16 The initial step of assigning the configuration of 1 involved derivatization of fragments I (2) and II (3), obtained by cleavage using NaIO4 and NaBH4, into their corresponding Mosher’s ester. More specifically, the six hydroxyl groups present in 2 were esterified using (R)- and (S)-MTPA-Cl. All proton chemical shifts of the two ester derivatives were assigned, and their corresponding differences (ΔδSR) were calculated. The configurations for the five secondary alcohol moieties were deduced from the ΔδSR signs for 1,3-diol systems as proposed by Riguera et al.17 As shown in Figure 3, the 1,3,5triol present in the C-6−C-10 segment could be considered as a superposition of two 1,3-diol units. According to the interpretation of Riguera, the sign distribution of the calculated ΔδSR values for C-6−C-10 was consistent with one case of

Figure 1. COSY, HMBC, and key HSQC-TOCSY correlations.

correlations with H-42 and H-46. Further observation of the DQF-COSY and TOCSY correlations with H-14 (δH 3.73) and H-18 (δH 3.74) suggested the linkage of C-14 and C-18 through a 2-hydroxypropyl moiety, which was confirmed by two heteronuclear multiple bond correlations (HMBC) between H-14/H-18 and the C-16 oxymethine carbon atom. Furthermore, both H-34 (δH 3.23) and H-38 (δH 3.28) were coupled with the proton(s) observed at δH 3.94 in the COSY spectrum, in addition to the two carbon atoms resonating at δC 38.0 and 72.6 in the HMBC spectrum. These observations suggested the presence of two oxymethine moieties with the same proton and carbon chemical shifts (δH 3.94/δC 72.6) in addition to a methylene group, thereby implying the presence of a 1,3-dihydroxypropyl unit. This unit therefore appeared to link carbon atoms C-34 and C-38 to give a relatively long linear chain from C-10 to C-47. Further connection with the two remaining oxymethine signals (δC 69.2/δH 3.80 and δC 66.4/δH 4.08) was confirmed through DQF-COSY and HSQC-TOCSY correlations with H-10. Comparison of the carbon chemical shifts of the two 1,3,5-triols in the C-14 to C-18 and C-29 to C33 chain indicated that the C-6 to C-10 carbon chain also corresponded to a 1,3,5-triol system. Moreover, compound 1 was found to possess two exomethylene groups, likely positioned at the termini of the carbon chain. From the observed COSY correlations, it is apparent that one of these groups is a monoene, while the other is a diene. In addition, the H-3 methylene protons, which were coupled with an olefinic proton, produced HSQC-TOCSY correlations with one of the terminal exo-methylene carbon atoms, two aliphatic

Figure 2. Fragments produced following cleavage using NaIO4 and NaBH4 and the corresponding MS spectrum for the fragment mixture. 196


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The subsequent stereogenic configuration was achieved through measurement of home- and heteronuclear coupling constants between the nonequivalent methylene protons on C17. Upon designation of the up- and downfield shifted protons as H-17a and H-17b, respectively, the measured home- and heteronuclear coupling values indicated an anti relationship between C-15/C-18 and H-17b/16-OH. Similarly, the approximate coupling constants 3 J H‑17a,H‑18 ∼ 3.0 Hz, 3 JH‑17b,H‑18 ∼ 9.2 Hz, 2JH‑17a,C‑18 ∼ − 1.1 Hz, and 2JH‑17b, C‑18 ∼ − 5.6 Hz were sufficient to assign the anti orientation of C-16/ C-19 and H-17a/18-OH. These two conformers resulted in an anti/anti relationship for 1,3,5-triol system in the C-14−C-18 segment, as observed for the configuration of the oxymethine carbon atoms in the C-6−C-10 chain. These proposed 1,3,5triol configurations were also supported by Kishi’s NMR database for a 1,3,5-triol moiety, where the chemical shift of the center carbon in a 1,3,5-(anti/anti)-triol is ∼66.3 ppm in CD3OD.18,19 For the C-18/C-19 bond, the four small homeand heteronuclear coupling constants of 3JH‑18,H‑19, 2JH‑18,C‑19, 2 JH‑19,C‑18, and 3JH‑18,C‑20 allowed the syn orientation to be assigned for 18-OH to 19-OH. The subsequent oxymethine carbon atom configuration was assigned in a similar manner as that employed for the C-16−C18 unit. More specifically, the JBCA of C-19/C-20 indicated anti orientations of C-18/C-21 and H-20b/19-OH, as previously determined for C-16/C-17. On the basis of this configuration, coupling constants of 3JH‑20a,H‑21 ∼ 8.0 Hz, 3 JH‑20b,H‑21 ∼ 3.9 Hz, 2JH‑20a,C‑21 ∼ − 5.4 Hz, and 2JH‑20b,C‑21 ∼ − 2.0 Hz led to assignment of the anti orientation for C-19/C-22 and H-20b/21-OH, thereby suggesting a 1,3-(syn) configuration between C-19 and C-21. A syn relationship was also supported in this case due to similarities in the chemical shift for C-21 and the corresponding carbon atom in Kish’s NMR database for a 1,3-diol system (i.e., 71.5 ppm in CD3OD).20−22 For the C-21/C-22 bond, the continuous JBCA defined the orientation of the two nonequivalent protons on C-22 as shown in Figure 4. In addition, based on the relative orientations of the two protons on C-22, the configuration of the tetrahydropyran ring was determined by ROE correlations (Figure 5). Thus, the

Figure 3. Mosher ester analysis of (a) fragment I (2) and (b) fragment II (3).

possible configurational combinations of two 1,3-diols. Accordingly, the configurations of C-6, C-8, and C-10 were assigned as 6S, 8R, and 10R, respectively. Similarly, the ΔδSR sign distribution of carbon atoms present in C-14−C-16 corresponded to one of the four configurations available for 1,3diol systems,17 and so the absolute configurations of C-14 and C-16 were assigned as 14R and 16R. On the basis of the stereochemical assignment of the 14R and 16R centers, the configurational assignments of C-13, C-18, C-19, and C-21 were successfully accomplished by application of the JBCA method. For this purpose, the homo- and heteronuclear coupling constants were measured from the obtained DQF-COSY and HECADE spectra, respectively. A small homonuclear coupling constant between H-13 and H-14 and a large heteronuclear coupling constant between H-14 and CH3 indicated that H-14 is placed in a gauche orientation with H-13 and in an anti position with respect to CH3 (Figure 4). In addition, the small heteronuclear coupling constants of H-13/ C-14 and H-13/C-15 are indicative of an anti orientation between H-13 and the hydroxyl group on C-14 but a gauche orientation between H-13 and C-15. From these four coupling constants, the configuration of C-13 was assigned as R.

Figure 4. J-based configuration analysis for the C-13−C-22 fragment. 197


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configurations of C-26, C-27, and C-28 (within the ring) to be assigned as 26R, 27S, and 28R, respectively. Following on from the above analyses, the configurations of the stereogenic centers present on the second chain linked to the tetrahydropyran ring could be established based on the stereochemical assignment of C-29, which was determined to adopt the S configuration based on the small coupling constant of H-28/H-29 (i.e., 3JH‑28,H‑29 ∼ 2 Hz) and ROE correlations between H-27/H-29 and H-28/H-30a. Unlike the previous cases, H-29 exhibited intermediate homonuclear coupling constants (i.e., 3−7 Hz) with the neighboring methylene protons, implying interconversion between two conformers in the JBCA method. This was supported by ROE observations over five bonds between H-28 and H-31 in one of the two conformers. The observed homo- and heteronuclear coupling constants between the subsequent proton and carbon atoms on the chain then allowed the anti orientation to be assigned to C29/C-32 and H-30a/31-OH and the gauche conformation to be assigned to H-30b/31-OH. The orientation between 29-OH and 31-OH was therefore established to be syn. Upon

Figure 5. Determination of the tetrahydropyran ring configuration using ROE correlations.

ROE correlations of H-22a/H-23a, H-22b/H-23b, and H-22a/ H-24, along with a 2JH‑23b,C‑24 value of 9.2 Hz and weak HMBC correlations between H-23a/C-24 and H-23a/C-25 indicated an R configuration for C-24. Further ROE correlations between H-23b/H-26, H-23b/H-28, and H-25a/H-27 allowed the

Figure 6. J-based configuration analysis for the C-28−C-43 fragment. 198


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Figure 7. Apoptosis of various cell lines in the presence of ostreol B (1) as determined using the Annexin-V/PI double staining assay.

by the presence of the two ROE correlations between H-33/H39 and H-36b at a distance of five bonds with a chemical shift of 2.21 ppm. Application of the JBCA method was then extended to determine the configurations of the C-35−C-37 and C-39−C41 units containing nonequivalent methylene protons. As shown in Figure 6, the magnitude and sign of the coupling constants of the C-35−C-36 bond were examined, and these values were found to be comparable to those of the C-36−C-37 bond, thereby indicating that the orientations of C-34/C-37, C35/C-38, 35-OH/H-36b, and 37-OH/H-36b are anti. In addition, the alternating conformers formed from the C-39− C-40 and C-40−C-41 units were deduced from the intermediate values of the homo- and heteronuclear coupling constants and were confirmed by the presence of ROE correlations between H-38/H-41 and H-39/H-42. Thus, based on the observed coupling values, 39-OH was assigned to be the syn orientation with respect to 41-OH. Moreover, for the C-41−C-42 bond, the observed small coupling constants also suggested syn orientation for 41-OH−42-OH. Interestingly, all hydroxyl groups present along the C-33 to C-42 chain adopted the syn orientation, and the carbon atoms were

comparison of the magnitude and sign of each homo- and heteronuclear coupling constants for C-31−C-32 and C-32−C33, an anti orientation was determined between 31-OH and H32b and between 33-OH and H-32a. These assignments indicated that the 1,3,5-triol system in the C-29−C-33 segment adopted a syn/anti relationship. This proposed configuration was also supported by minimal differences in the carbon chemical shifts between C-31 and the middle oxymethine carbon in the 1,3,5-triol of Kishi’s NMR database (i.e., 68.4 or 68.2 ppm in CD 3 OD) in the syn/anti or anti/syn configurations.18,19 Application of the JBCA method to the subsequent proton and carbon centers suggested the syn orientation for C-33−C34 based on the small coupling constants, as was the case for C18−C-19. Similarly, it was determined from their small homoand heteronuclear coupling constants that C-38/C-39 and C41/C-42 constitute 1,2-diol systems in the syn orientation. Moreover, although the homo- and two-bond heteronuclear coupling constants for the C-34−C-35 and C-37−C-38 units were intermediate, thereby indicating interconverting conformations, the configuration for these 1,2-diols was also assigned as syn. These conformations were further confirmed 199


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through a combination of JBCA, ROE correlations, and the modified Mosher method. Furthermore, in the case of the 1,3,5-triol system, the chemical shift of the center carbon atom was compared with that reported in Kishi’s NMR database to confirm the configuration. Moreover, ROE correlations through five bonds distance confirmed the presence of interconverting conformers, as indicated by the JBCA method. Additionally, the similar homo and heteronuclear coupling constants in two pairs of conformers (i.e., C-19−C-20 and C-35−C-36) confirmed that the two 1,3-diol moieties adopted identical configurations. Following structural characterization of 1, we also confirmed its cytotoxicity against a range of cancer cells, with low IC50 values being calculated in all cases. We expect that these results will be of particular interest, as they could be used to confirm the structural and conformations of other products, and the techniques employed herein could be applied in the structural determination of similar compound from O. cf. ovata. Finally, the attempted isolation of other compounds from cytotoxic O. cf. ovata fraction produced additional linear polyhydroxy-containing compounds that differed in structure from osterol B. Determination of the chemical structures of these compounds will be carried out in the near future, and the results will be presented in due course.

assigned the following configurations: 33S, 34R, 35R, 37S, 38S, 39R, 41S, and 42S. Accordingly, overlap between the 13C NMR signals corresponding to C-35 and C-37 was likely due to the high symmetry within this segment. The configuration of the isolated stereogenic C-46 center was then established independently to be S, using the Mosher method for fragment II (3); the sign distribution of ΔδSR for the protons surrounding the C-46 atoms in the two MTPA esters is shown in Figure 2. As indicated, C-46 adopts the syn configuration toward C-42, thereby rendering the fraction a 1,5(syn)-diol. This configuration is supported by the nonequivalent chemical shifts (δH 1.39 and 1.64) of H-44, which corresponds with Matsunaga’s empirical rule16 (i.e., two oxygen atoms in a 1,5-(syn)-diol system can influence the center methylene protons in an nonequivalent manner), while two oxygen atoms in a 1,5-(anti)-diol provide symmetrical shielding to the center protons. Finally, based on the rather large proton coupling constant (3JHH = 15.4 Hz) and ROE signal of H-53/ H-55, the Δ53,55 double bonds were found to adopt the all E geometry. These results allowed us to conclude that ostreol B (1) possesses numerous hydroxyl groups along the two long carbon chains that extend from the tetrahydropyran ring and terminate with exo-methylene moieties. Furthermore, all configurations in 1 were successfully determined by the combination of diverse NMR methods and chemical reactions, and the chemical shifts of 1,3-diol, 1,5-diol, and 1,3,5-triol moieties were confirmed by Kishi’s NMR database and 1,5-diol empirical rule. We then evaluated the in vitro cytotoxicity of ostreol B in hepatocarcinoma (HepG2), neuroblastoma (Neuro-2a), and colon cancer (HCT-116) cell lines. 1 exhibited moderate cytotoxicity against all cell lines, with IC50 values of 4.8, 0.1, and 0.9 μM being calculated for the HepG2, Neuro-2a, and HCT116 cells, respectively. In addition, apoptosis induced by ostreol B at the concentrations of 10 and 20 μM was examined using the Annexin-V/propidium iodide (PI) double staining assay (Figure 7).23,24 Figure 7 shows the distribution of nonapoptotic (bottom left), early apoptotic (bottom right), late-apoptotic (top right), and dead cells (top left) following Annexin V/PI staining analysis for cells treated with ostreol B for 24 h. As indicated, ostreol B induced an increase in the population of apoptotic or dead cells in all cell lines tested. In HepG2 cells, no significant change in the percent of cells undergoing apoptosis was observed after 24 h incubation at 10 μM ostreol B, whereas the percentage of late apoptotic cells (Annexin-V+/ PI+) increased to 19.05% at 20 μM. In neuro-2a cells, the population of late apoptotic cells (Annexin-V+/PI+) was significantly increased at both 10 and 20 μM. In HCT-116 cells, the increase of dead cells (Annexin-V−/PI−) was relatively remarkable after 24 h to 65.70 and 55.15% at 10 and 20 μM, respectively.

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EXPERIMENTAL SECTION

Instrumentation. Optical rotation measurement was carried out on a JASCO P-1010 polarimeter with a 5 cm cell. IR spectrum was recorded on a JASCO FT/IR 4100 spectrometer. All NMR spectra were measured on a Bruker AVANCE II 900 MHz spectrometer in CD3OD (residual solvent peaks at δH 3.30 ppm), and high-resolution ESI TOF mass spectrum was acquired using a Waters SYNAPT G2 system. The NMR and HRMS data were supplied by the Korea Basic Science Institute, Ochang Center, Korea. ESI-MS/MS spectra were acquired on an AB SCIEX QTRAP 3200 LC/MS/MS system, and HPLC was performed using an Agilent 1200 system and a Spectra Physics P2000 pump equipped with a Waters UV480 detector and a YMC ODS-A column. All solvents were distilled prior to use. Microalgal Material. The isolation and culture of the dinoflagellate Ostreopsis cf. ovata was described in our previous publication.7 Isolation and Purification of 1. All cytotoxic fractions obtained in our previous study were combined and subjected to fractionation using a Sephadex LH-20 open column to yield five subfractions (i.e., I−V). Among these subfractions, subfraction II (∼60 mg), which exhibited cytotoxicity in the brine shrimp lethality bioassay, was further separated by reversed-phase HPLC (YMC ODS-A, 250 × 10 mm, id, 235 nm UV detector) eluting with a solvent mixture composed of water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B) at a flow rate of 2 mL/min using the following solvent step gradient: 0−2 min (20% B); 2−5 min (20−30% B); 5−20 min (30−40% B); 20−30 min (40−50% B); 30− 35 min (50−60% B); 35−40 min (60−70% B); 40−50 min (70− 100% B), and 50−55 min (100% B) to obtain mixture of cytotoxic compounds at a retention time of 32 min. This mixture was further purified by reversed-phase HPLC using a YMC-C18 semipreparative column and an eluting solvent of 75% aqueous MeOH containing 0.1% formic acid to yield ostreol B (1, 2.6 mg) at a retention time of 30 min. Ostreol B (1). Colorless amorphous solid; [α]24D + 2.7 (c 0.21, MeOH); IR (film) 3343, 1058 cm−1; UV (MeOH) λmax 235 (ε 19,400) nm; HRESI-MS m/z 1167.7227 [M + Na]+ (calcd for C57H108O22Na, 1167.7230). Full 1H and 13C NMR data can be found in Table 1. Oxidative Cleavage of 1 with NaIO4. Sodium iodate (10 mg) was added to a solution of 1 (2.0 mg) in acetone (1 mL) and water (0.4 mL), and the mixture was stirred at room temperature for 1 h. After this time, the mixture was diluted with ethyl acetate (EtOAc) and

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CONCLUSION Following the isolation of polyhydroxy compound ostreol B(1) from cultivated cells of the toxic dinoflagellate O. cf. ovata, a number of 1D and 2D NMR spectroscopic techniques were successfully employed to determine the planar chemical structure of this cytotoxic compound. More specifically, this structure was found to contain a central tetrahydropyran ring, two terminal exo-methylene moieties, and 21 hydroxyl groups, which were present in the form of 1,2-diol, 1,3-diol, and 1,3,5triol moieties. The absolute configurations of the various stereogenic centers and diol/triol groups were determined 200


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The Journal of Organic Chemistry partitioned by the addition of water. The organic fraction was then dried and subjected to the reversed-phase silica HPLC using a semipreparative YMC-A column and an eluting solvent containing 60% aqueous MeOH to give fragments I (2) (0.5 mg) and III (4) at retention times of 19 and 8 min, respectively. Fragment II (3) (0.4 mg) was obtained following column washing. Preparation of the (S)- and (R)-MTPA Esters of Fragments I (2) and II (3). Fragment I (2) (0.5 mg) was separated into two vials in equal portions (0.25 mg amount) and 4-dimethylamino pyridine (0.4 mg), and anhydrous pyridine solvent (20 μL) were added to each vial. After heating the vials to 40 °C with stirring, R(−)-MTPA-Cl (20 μL) was added to one of the vials, and stirring continued for 2 d. The reaction mixture was then quenched by the addition of water and dimethyl ether, the organic solvent was evaporated in vacuo, the mixture was purified by HPLC (silica gel, 70%:30% EtOAc/hexane) to give the (S)-MTPA-ester of 2 (tR ∼ 8 min). 1H NMR (900 MHz, CDCl3): 5.64 (HI-1), 4.96 (HI-16), 4.94 (HI-8), 4.93 (HI-2), 4.89 (HI6), 4.87 (HI-14), 4.82 (HI-10), 4.17 (HI-18b), 4.12 (HI-18a), 1.96 (HI17b), 1.92 (HI-17a), 1.91 (HI-7b), 1.90 (HI-3), 1.88 (HI-7a), 1.82 (HI9b), 1.74 (HI-9a), 1.68 (HI-15), 1.63 (HI-13), 1.52 (HI-11b), 1.51 (HI5b), 1.45 (HI-5a), 1.44 (HI-11a), 1.22 (HI-4), 1.14 (HI-12b), 0.91 (HI12a), 0.60 (HI-CH3); [M + 2Na]2+ = 853. Similarly, the (R)-MTPA-ester of 2 was obtained using S(+)-MTPA-Cl after reaction in the second vial over 1 d: 1H NMR (900 MHz, CDCl3): 5.68 (HI-1), 4.96 (HI-8), 4.94 (HI-2), 4.90 (HI16), 4.88 (HI-6), 4.84 (HI-10), 4.83 (HI-14), 4.23 (HI-18b), 4.06 (HI18a), 1.95 (HI-17b), 1.94 (HI-3), 1.88 (HI-17a), 1.86 (HI-7), 1.84 (HI9b), 1.78 (HI-9a), 1.64 (HI-15b), 1.61 (HI-13), 1.60 (HI-5b), 1.56 (HI15a), 1.51 (HI-11b), 1.50 (HI-5a), 1.33 (HI-11a), 1.28 (HI-4), 1.05 (HI-12b), 0.82 (HI-12a), 0.69 (HI-CH3); [M + 2Na]2+ = 853. Fragment II (3) (0.4 mg) was separated into the two vials in equal portions (0.20 mg), and derivatization was carried out as described above using R(−)-MTPA-Cl (20 μL) over 4 h. Purification was by HPLC (silica gel, 70:30 EtOAc/hexane) to give the (S)-MTPA-ester of 3 (tR ∼ 10 min). 1H NMR (900 MHz, CDCl3): 5.03 (HII-11), 4.29 (HII-15b), 4.27 (HII-15a), 1.68 (HII-14), 1.64 (HII-12b), 1.58 (HII12a), 1.51 (HII-10), 1.33 (HII-13), 1.18 (HII-9); [M + Na]+ = 696. Similarly, the (R)-MTPA-ester of 3 was obtained using S(+)-MTPA-Cl after reaction for 3 d: 1H NMR (900 MHz, CDCl3): 5.03 (HII-11), 4.21 (HII-15b), 4.19 (HII-15a), 1.60 (HII-10, −11b, −14), 1.55 (HII-12a), 1.27 (HII-13), 1.26 HII-9); [M + Na]+ = 696. Estimation of Cytotoxicity. HCT-116 (human colon cancer cells) and HepG2 (human liver carcinoma cells) were obtained from the Korean Cell Line Bank (Seoul, South Korea). Neuro-2a (mouse brain neuroblastoma cells) was purchased from ATCC (Manassas, VA). All cells were maintained in Dulbecco’s Modified Eagle’s medium containing 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (10 mg/mL) at 37 °C in an atmosphere of 5% CO2 atmosphere and 95% relative humidity. All compounds of interest were dissolved in DMSO (final concentration, 0.05%) and diluted in serum-free culture medium. Prior to carrying out the assay, the cells were seeded and incubated for 24 h in 48-well plates (HCT-116: 1 × 105 cells/mL; neuro-2a cells: 2 × 105 cells/mL; HepG2 cell: 5 × 104 cells/mL; 100 μL per well). The cells were then treated with the desired vehicle or the compound of interest at the specified concentration (as indicated above) over 48 h. The inhibitory activity of each compound toward cell proliferation was assessed using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, by incubation of the cells with 2 mg/mL MTT for 2 h. After this time, the supernatant was aspirated and DMSO (200 μL) was added to dissolve the produced formazan dye. The absorbance of the resulting solution was then measured at 450 nm using a microplate reader. The data were expressed as percentage of viable cells relative to the vehicle-treated control cultures and reflect average values from three independent experiments. The IC50 values refer to the concentrations at which 50% inhibition was achieved and were calculated from regression plots based on a minimum of five different concentrations. Apoptosis Determination using Flow Cytometry. To measure cell apoptosis, HCT-116, HepG2, and Neuro-2a cells were seeded in

12-well plates and treated with ostreol B at concentrations of 10 and 20 μM. After 24 h, the cells were collected, washed with phosphatebuffered saline, and subjected to centrifugation at room temperature. To determine the degree of cell apoptosis, cell pellets were prepared using a Muse Annexin V and Dead Cell Assay kit (EMD Millipore Cop., MA) according to the manufacturer’s manual. Apoptosis of each cell line was evaluated using flow cytometry (Muse Cell Analyzer).25

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b02569. HR-ESIMS and MS/MS data of 1; 1D and 2D NMR spectra for 1, and 1H NMR spectra for (S)/(R)-MTPA esters of 2 and 3 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82 63 469 4606. ORCID

Jung-Rae Rho: 0000-0001-6443-632X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Research Foundation (NRF) grant by the Korea government (MSIP) (NRF-2015M1A5A1041808). The NMR experiments were supported by the Korea Basic Science Institute under the R&D program (Project D37700) supervised by the Ministry of Science and ICT.

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REFERENCES

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